The present invention relates to compositions which are useful in potentiating neurotrophin activity, as well as methods for the preparation and use thereof.
Protein growth factors of the neurotrophin family, which includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and neurotrophin-5 (NT-5) regulate nervous system development [Barde, Y-A., "Trophic factors and neuronal survival," Neuron 2:1525-1534 (1989); Thoenen, H., "The changing scene of neurotrophic factors," Trends Neurosci 14:165-170 (1991); Leibrock, J. et al., "Molecular cloning and expression of brain-derived neurotrophic factor," Nature 341:149-152 (1989); Ernfors, P. et al., "Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family," Neuron 5:511-526 (1990); Hohn, A. et al., "Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family," Nature 344:339-341 (1990); Maisonpierre, P. C. et al., "Neurotrophin-3: a neurotrophic factor related to NGF and BDNF," Science 247:1446-1451 (1990); Rosenthal, A. et al., "Primary Structure and Biological Activity of a Novel Human Neurotrophic Factor," Neuron 4:767-773 (1990); Jones, K. R. and Reichardt, L. F., "Molecular cloning of a human gene that is a member of the nerve growth factor family", Proc. Natl. Acad. Sci. USA 87:8060-8064 (1990); Hallbook, F. et al., "Evolutionary studies on the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary," Neuron 6:845-858 (1991); Berkemeier, L. R. et al., "Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB," Neuron (in press)]. In addition, the neurotrophins are strongly implicated as playing an important role in structural maintenance, plasticity and repair of the adult nervous system [Hefti, F. et al., "Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerative diseases," Neurobiol. Aging 10:515-533 (1989)].
Neurobiological research carried out in recent years has confirmed that development, maintenance of function and regeneration of neurons is profoundly influenced by the neurotrophic factors. These neurotrophins stimulate mechanisms necessary for survival, neurite growth and functions related to transmitter production and release. For example, it has long been known that nerve growth factor (NGF), the first and best characterized neurotrophin, is a neurotrophic factor for peripheral sympathetic and sensory neurons, and more recent findings show that NGF also affects cholinergic neurons in the brain. NGF is required by sympathetic and dorsal root ganglion cells for survival during embryonic and early postnatal life, and is also critical to the normal function of these neuronal types in adult animals. NGF is further implicated in the regulation of a variety of developmental processes such as naturally-occurring cell death, differentiation, process outgrowth and synaptic rearrangement.
Experiments over the last few decades have yielded evidence that NGF regulates a variety of cellular processes important for neuronal function. Administration of pharmacological doses of NGF to rodents results in striking increases in ganglion cell size, axonal branching in the periphery and dendritic arborization as demonstrated by, e.g., intracellular staining techniques. Furthermore, administration of NGF leads to increases in the synthesis of transmitter enzymes and increases in the synthesis of peptides in dorsal root ganglion cells. NGF also exerts effects on preganglionic neurons innervating sympathetic ganglion cells, presumably an indirect effect of its influence on the ganglion cells. Importantly, NGF can prevent death of responsive neurons pursuant to mechanical, chemical and immunological insults. When NGF deprivation is induced by axotomy or administration of antisera, atrophy and reduction in the synthesis of transmitter enzymes occur. Furthermore, when autoimmunity to NGF is induced in rats, guinea pigs and rabbits, there is massive death of sympathetic ganglion cells over a period of several months in animals that generate high antibody titers. Finally, even in adulthood, several neuronal populations in the peripheral and central nervous system respond to transection of their axons by atrophy, reductions in transmitter synthesis and significant degrees of cell death. Taken together, all of these finding in vivo suggest that trophic factors act chronically in the mature animal to maintain normal function. Therefore, trophic deficiency is probably an important mechanism in disease states of adulthood [see Snider, W. D. and Johnson, Jr., E. M., "Neurotrophic Molecules," Annals of Neurology 26:489-506 (1989) and references cited therein].
Other neurotrophic molecules characterized thus far influence various other neuronal populations. The existence of different patterns of specificity suggests that there may be a multitude of neurotrophins with different specificities and activities. As the molecules occur in minimal quantities, their isolation is a cumbersome and time-consuming effort.
The discovery of neurotrophic factors has obvious implications with respect to neurodegenerative diseases. Indeed, it has been hypothesized that the lack of neurotrophic factors is responsible for the degeneration of selective neuronal populations as it occurs in Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, and that application of corresponding neurotrophic factor might prevent neuronal degeneration [Appel, S. H., "A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism, and Alzheimer's disease," Ann. Neurol. 10:499-505 (1981)]. In particular, as NGF is a trophic factor for the population of basal forebrain cholinergic neurons which degenerates in Alzheimer's disease, it has been speculated that NGF may be useful in the treatment of this disease.
Classical neuropharmacology attempts to influence mechanisms related to neuronal impulse flow and transmission at the synapse. Currently-used drugs and available pharmacological tools do not affect the structural features of the central nervous system. Moreover, there is a lack of compounds that are able to promote regeneration, plasticity and maintenance of structural integrity of selected neuronal systems. An increased understanding of the properties of neurotrophic factors is virtually certain to lead to the development of a new, structurally-oriented neuropharmacology. In particular, neurotrophic factors shall undoubtedly prove useful in the treatment of neurodegenerative diseases associated with structural disintegration of selected neuronal systems of brain areas.
One of the more exciting features of neurotrophic molecules from a clinical standpoint is their ability to promote cell survival after a variety of insults. For example, it has been shown that NGF has the ability to save neurons that would ordinarily die after mechanical injury. These injuries have been most commonly produced by transecting the axons of sympathetic and dorsal root ganglion cells or basal cholinergic forebrain neurons. Such injuries separate the soma from contact with targets and presumably cause neurons to degenerate because of loss of trophic support, although other mechanisms may be involved. In every circumstance in which axons of a responsive neuronal population have been transected, NGF has saved at least some neurons from degenerating. NGF works after systemic administration for peripheral neurons, as well as after local application to axon tips, and is effective after intraventricular administration for neurons within the central nervous system. Another neurotrophic molecule, FGF, is also effective in some of these same paradigms. This ability to prevent cell death after injury is obviously relevant to the problem of promoting neural regeneration [see Snider and Johnson, supra, at 498 et seq.].
Neurotrophins have also been shown to save neurons after exposure to certain toxins. For example, the concomitant administration of NGF with 6-hydroxydopamine (which is presumed to act by destroying sympathetic nerve terminals, thereby interfering with the uptake of NGF from a target) can completely prevent the death of cells that occurs upon administration in early postnatal life. In addition, NGF has been shown to save neurons after administration of vinblastine and coichicine, which inhibit axoplasmic transport. Further, NGF can partially prevent the cell death in dorsal root ganglia caused by administration of the sensory toxin capsaicin to newborn animals.
Neurotrophins are also implicated in a number of different ways with an organism's maintenance of healthy neuronal function. For example, NGF has been shown to suppress primary infection of dorsal root and sympathetic ganglion cells by herpes simplex type I virus; NGF is believed to suppress the expression of gene products necessary for viral replication. In addition, lymphocytic infiltration and destruction of sympathetic ganglia induced by administration of guanethidine and its analogues (resulting in autoimmune attack) is completely prevented by concomitant administration of NGF. The presumed mode of action is by suppressing the expression of the antigen on the ganglion cell surface, leading to the suggestion that another physiological role of trophic factors in adult animals may be to maintain immunological silence of irreplaceable neurons [Snider et al., supra, at 499].
Unfortunately, several formidable obstacles remain to be overcome before neurotrophic peptides can be of widespread clinical utility. First, sufficient quantities of the neurotrophins must be available; recombinant DNA technology will be required to engineer expression vectors that produce large quantities of biologically active factors. Further, the practical difficulties of and limitations on the administration and delivery of such molecules must be overcome. In order to be able to reach neuronal populations in the brain, neurotrophic factors would have to be given intracerebrally, as these proteins do not cross the blood-brain barrier. In human patients in particular, there would only be limited options for administration of neurotrophins per se. Neurotrophic factors purified from natural sources or produced by recombinant techniques could potentially be chronically infused into the brain with the help of mechanical pump devices; however, subcutaneous pumps are relatively complex devices necessitating surgical intervention, and stability of the active proteins during storage in these pump devices would be expected to necessitate special preparations. It is encouraging that local administration of NGF to the distal parts of injured neurons enhances survival and regeneration, thus suggesting the potential in some situations for local administration; nonetheless, even in those instances, the use of some sort of prosthetic device appears necessary. An alternative method of administration would involve the use of slow-release intracerebral implants containing the active protein embedded in a biodegradable polymer matrix. At this time, existing polymers provide stable release rates of only several weeks.
While administration of modified neurotrophin molecules or active fragments thereof may ultimately provide a solution to the problem of providing therapeutic agents with neurotrophic activities, at this time very little is known about the possibility of producing active fragments of neurotrophic factors. Accordingly, it has been suggested that perhaps the best long-range hope for this class of agents lies in understanding in detail their interaction with their receptors and the molecular mechanisms of their trophic and survival-promoting actions; this may allow the design and use of low-molecular-weight drugs that mimic the effects of trophic factors and that can be administered in more traditional and practical ways [Snider, supra, at 499]. The identification of agents that modify components of neurotrophin activity is in and of itself a valuable contribution to the art, in view of the substantial present utility of such agents in obtaining a clearer understanding of the molecular mechanisms involved and in the design of novel therapeutic agents.
At least two types of proteins are apparently involved in the formation of functional receptors for neurotrophin growth factors. These are the low affinity NGF receptor protein (p75-NGFR) [Chao, M. V. et al., "Gene transfer and molecular cloning of the human NGF receptor", Science 232:518-521 (1986); Radeke, M. J., et al., "Gene transfer and molecular cloning of the rat nerve growth factor receptor: a new class of receptors"; Nature 325:593-597 (1987)] and products of trk-related proto-oncogenes [Hempstead, B. L. et al., "High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor," Nature 344:339-341 (1990)]. The trk gene products, but not the p75-NGFR, exhibit protein kinase activity. Individual trk receptors bind to and stimulate tyrosine phosphorylation of different subsets of neurotrophins. Trk binds to NGF but not BDNF, trkB binds BDNF but not NGF. NT-3 is capable of interacting with trk and trkB receptors and with trkC. The interaction of NT-3 with multiple trk receptors may allow this factor to control the survival of populations of neurons expressing different trk gene products.
U.S. Pat. No. 4,555,402 to Matsuda et al., the entire disclosure of which is hereby incorporated by reference, discloses the isolation of a physiologically-active substance denominated as K-252. This compound is described as having antiallergic and antihistamine-releasing activities.
U.S. Pat. No. 4,923,986 to Murakata et al., the entire disclosure of which is also hereby incorporated by reference, discloses a class of derivatives of K-252 represented by the general formula ##STR1##
wherein W.sub.1, W.sub.2, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X and Y represent various substituents. The compounds are physiologically active substances that inhibit protein kinase C and exhibit an antitumor activity.
U.S. Pat. No. 4,877,776 to Murakata et al., the entire disclosure of which is also hereby incorporated by reference, discloses another class of derivatives of K-252 represented by the general formula ##STR2##
wherein R.sup.1 and R.sup.2 are independently H or OH, X represents COOH, COOR or CH.sub.2 OH; Y represents H, R or COR; and Z represents OH, OR or SR, in which R represents lower alky. These derivatives are described as exhibiting C-kinase inhibitory activity, and were expected to be useful as an active ingredient of antitumor agents, etc.
Of the compounds disclosed in the aforementioned U.S. patents, two have been known for the longest time and have in particular become the subjects of substantial research scrutiny. K-252a and K-252b, two related alkaloid-like compounds from microbial origin known to interfere with protein kinase activities in cell-free systems, have been found to inhibit several biological actions of NGF [Nakanishi, S. et al., "K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase," J. Biol. Chem. 263:6215-6219 (1986); Kase, H. et al., "K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases," Biochem. Biophys. Res. Commun. 142:436440 (1987); Koizumi, S. et al., "K-252a: a specific inhibitor of the action of nerve growth factor on PC12 cells," J. Neurosci. 8:715-721 (1988); Matsuda, Y. and Fukuda, J., "Inhibition by K-252a, a new inhibitor of protein kinase, of nerve growth factor-induced neurite outgrowth of chick embryo dorsal root ganglion cells," Neurosci. Lett. 87:295-301 (1989)]. K-252a prevents the NGF induced morphological transformation of proliferating PC12 pheochromocytoma cells into neuron-like cells and inhibits the NGF stimulated, but not the basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) stimulated phosphorylation of selected proteins [Hashimoto, S., "K-252a, a potent protein kinase inhibitor, blocks nerve growth factor-induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells," J. Cell. Biol. 107:1531-1539 (1988); Sano, M. et al., "A nerve growth factor-dependent protein kinase that phosphorylates microtubule-associated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells," J. Neurochem. 55:427-435 (1990)]. Thus, to date compounds as described in the aforementioned U.S. patents have been shown to have only an inhibitory affect on neurotrophin activity.
The development of non-peptide agonistic molecules for neurotrophic factors which pass the blood-brain barrier, while acknowledged as theoretically possible, has heretofore been considered potentially to prove a herculean task [Hefti, F. et al. (1989), supra, at 525]. It would therefore be highly desirable to identify low-molecular-weight agents which modulate (and, most desirably, potentiate) neurotrophin activity, both for purposes of elucidating the molecular mechanisms of neurotrophin action and as useful therapeutic agents for treatment of, e.g., neurodegenerative diseases.
It is an object of the present invention to provide compositions for use in potentiation of neurotrophin activity, as well as methods for the preparation and use thereof.